The amphetamine analogs of methylenedioxyphenylalkylamines are a series of compounds often referred to as designer amphetamines. These psychotropic drugs are ring-substituted derivatives chemically related to mescaline. They include 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy), 3,4-methylenedioxyethamphetamine (MDE, Eve), 3,4-methylenedioxyethylamphetamine (MDEA), 3,4-methylenedioxy-N-propylamphetamine (MDPA), 3,4-methylenedioxy-N-methylbutanamine (MBDB), and 3,4-methylenedioxybutanamine (BDB), the most common of these being MDMA.
The abuse of these designer amphetamines is increasing throughout the world, and their detection by screening methods is becoming a more important issue. Zhao, H. et al., J. Anal. Toxicology, Vol. 25, pp. 258-269 (2001) found 71% of urine samples from rave party attendees contained MDMA or MDA alone or in combination with amphetamine or other designer amphetamines such as MDEA.
Gas chromatography/mass spectrometry (GC/MS) is highly specific and has been described for the simultaneous detection of MDMA, MDA, amphetamine, methamphetamine, MDEA and their metabolites. GC/MS analysis is usually required for confirmation and verification of the results of an immunological assay or a suspected diagnosis. In this technique, MDMA or designer drugs are extracted in solid phase, then derivatized and analyzed via GC/MS. However, some medical facilities may not be able to detect ecstasy-class drugs because they lack the sophisticated and expensive instrumentation required.
In testing for drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as β-galactosidase and peroxidase, fluorescent molecules such as fluorescein compounds, radioactive compounds such as 125I, and microparticles are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
Until recently, there were no commercial immunoassays designed specifically for the detection of ecstasy-class drugs, and their detection therefore depended upon the relative cross-reactivities they exhibit in the amphetamine or methamphetamine screening method used. In general, the cross-reactivity of the commercially available amphetamine and methamphetamine assays toward many of these compounds is low, which means that the assays fail to detect ecstasy-class compounds at lower concentrations, which suggests the possibility that some positive samples may go undetected. Moreover, existing immunoassays for amphetamine and methamphetamine are limited by their cross-reactivity to allergy and cold medications such as ephedrine, pseudoephedrine and phenylpropanolamine, and to diet drugs such as phentermine. This cross-reactivity factor prevents the cut-off level for detection of amphetamine and methamphetamine from being lowered, which in turn, prevents detecting ecstasy-class drugs at lower concentrations. Therefore, an assay with increased specificity for ecstasy-class compounds is needed, either as an assay to detect ecstasy-class compounds alone or as an assay to detect ecstasy-class compounds as well as amphetamine and methamphetamine.
Amphetamine and methamphetamine antibodies with significant cross-reactivity to one or more members of the ecstasy class of drugs are well known (Cody, J., J. Anal. Toxicology 14:321, 1990).
The synthesis of substituted methylenedioxy (MDO) leashed aromatic compounds are known in the literature (European Patent Application 329,326, published Aug. 23, 1989).
UK Patent Application 2,361,473, published Oct. 24, 2001, describes ecstasy-class analogs for detection of ecstasy-class compounds in biological samples. The conjugates and immunogens described are derived out of the nitrogen position of MDA. The present application describes an alternate solution to the problem, i.e., conjugates and immunogens derived out of the carbon position of the MDO moiety of ecstasy-class compounds.